1. Field of the Invention
This invention relates to xanthine derivatives and to nephelometric inhibition immunoassays and kits wherein they can be employed.
2. Description of the Prior Art
Nephelometry involves the detection of light scattered or reflected toward a detector that is not in the direct path of the transmitted light (1).
The basic principles governing nephelometric inhibition immunoassay (NIIA) were reported over 40 years ago by Pauling et al. (2,3) and examined again in detail by Pressman (4). These authors proved that one could quantify small amounts of hapten (molecules of less than 4000 M.sub.r) by measuring chemically the decrease in the amount of precipitate formed from the interaction of hapten-specific antisera with polyhaptenic substances or conjugates of hapten with protein. The magnitude of the inhibition was best explained by the preferential interaction of the small quantity of hapten with antibodies of high avidity. The measurement of hapten by NIIA in which an endpoint nephelometer is used to measure the light scattered by reaction between a specific antibody and a hapten-macromolecular conjugate has been reported (5,6). Nishikawa et al. advanced the method into the area of therapeutic drug monitoring by describing similar endpoint NIIAs for phenytoin, phenobarbital, and theophylline (7,8).
Nephelometric procedures are a convenient tool for monitoring antigen-antibody reactions at an early stage, by detecting the growth of complexes ("scattering centers") capable of scattering light before they separate out of solution as immunoprecipitates. The formation of these scattering centers can be accelerated by the use of hydrophilic non-ionic polymers (e.g., dextran, polyethylene glycol), which increases the probability of protein-protein interaction by excluding a significant fraction of water. The use of polymers in an immunonephelometric assay also gives the advantages of increased sensitivity and less antiserum consumption (9).
The hapten of interest (a substance that can react with an antibody but cannot cause an immunological response) is covalently linked to a carrier protein, and the resulting conjugate is used to immunize animals. The specific antiserum is then reacted with a second conjugate or developer antigen, such that several hapten molecules are bound to each molecule of an unrelated carried protein. Therefore, although haptens cannot be quantitated by direct nephelometric procedures, by taking advantage of the fact that haptens will form soluble immune complexes, one can develop assays in which the hapten inhibits the formation of light-scattering centers produced by reacting a developer antigen with a limited amount of specific antibody.
Rate NIIAs have also been reported (10,11).
Nephelometric inhibition immunoassays as a whole also possess those advantages characteristic of all homogenous immunoassays (e.g., enzyme multiplied immunoassay and substrate-labeled fluorescence immunoassay), namely, increased accuracy and precision because of the elimination of a separation step (which is common to all heterogeneous immunoassays radioimmunoassays, enzyme-linked immunosorbent assay, etc.). In addition, NIIAs, which are readily adaptable to automation, involve extremely stable reagents, compared with assays that require radioactive or enzymelabeled tags, for which shelf life is a constant problem.
In the prior art there exists a nephelometric assay and kit for theophylline which employs a theophylline-8-butyric acid derivative having the following formula: ##STR1## wherein n is the number of xanthine derivatives bonded to apoferritin. The cross-reactivity of the theophylline antiserum against major drugs and drug metabolites, i.e., the concentrations of cross-reactants in micrograms (.mu.g) per milliliter (mL) required to produce a 30% error at a theophylline concentration of 10 .mu.g/mL is set forth in Table I.
TABLE I ______________________________________ CROSS REACTIVITY OF THEOPHYLLINE ANTISERA Concentration .mu.g/mL Producing a 30% Error Compound at Theoophylline 10 .mu.g/mL ______________________________________ Caffeine &gt;200 Theobromine &gt;100 1,7-Dimethylxanthine &gt;250 1-Methylxanthine &gt;100 3-Methylxanthine &gt;100 7-Methylxanthine &gt;100 1,3,7-Trimethyl Uric Acid &gt;250 1,3-Dimethyl Uric Acid &gt;40 1-Methyl Uric Acid &gt;200 Uric Acid &gt;200 3-Methyl Uric Acid &gt;100 Xanthine &gt;200 Hypoxanthine &gt;100 8-Chlorotheophylline &gt;45 Diphenhydramine &gt;200 Diphylline &gt;100 Aminophylline &gt;2.5 ______________________________________
The correlation of this prior art theophylline assay kit with Syva Company's EMIT brand theophylline assay kit on a Gilford Instruments Model 203-S spectrophotometer is set forth in Table II.
TABLE II ______________________________________ COMPARISON OF PRIOR ART THEOPHYLLINE TEST AND REFERENCE METHOD Least Squares Correlation Reference Method (X) N Regression Equation Coefficient ______________________________________ Syva/Gilford 203-S 83 y = 1.01X + 0.361 0.972 ______________________________________
Although this prior art theophylline assay kit possesses a very low cross-reactivity and an excellent correlation with a reference method, it is very temperature sensitive. For example, the least square regression equations for observed (Y) versus expected (X) theophylline concentrations obtained at three different environmental (reaction) temperatures is set forth in Table III:
TABLE III ______________________________________ TEMPERATURE SENSITIVITY STUDY Least Squares Regression Temperature, .degree.C. Equation ______________________________________ 18 Y = 1.7663X - 6.9895 25 Y = 0.9211X + 1.1684 30 Y = 0.8971X + 3.0655 ______________________________________
Accordingly, it would be very desirable to have a NIIA and kit for use therein which also exhibits a low crossreactivity and an excellent correlation with a reference method, but in addition thereto is less sensitive to environmental temperature changes.